The present invention relates to a yeast transformation tool or cassette family leaving in yeast no exogenous DNA fragment other than the fragments coding for proteins of interest.
The subject of the present invention is an integration-excision cassette which makes it possible to inactivate one or more alleles of a gene and/or to insert a new gene, leaving in the host strain only yeast DNA and possibly the new gene.
The present invention also relates to a high copy plasmid in yeast having no unnecessary or useless exogenous DNA fragment, i.e. no exogenous DNA that is not required for the yeast searched function.
The invention also relates to a method of transforming yeasts using the said tools or cassettes as well as the transformed strains obtained.
Improving the productivity and robustness of yeast strains is a constant concern to which recombinant DNA technologies may help to provide answers.
Gene replacement is a molecular biology technique which is frequently used in yeast. A DNA fragment is cloned into a vector and then it is introduced into the cell to be transformed. The DNA fragment integrates by homologous recombination into a targeted region of the recipient genome (Orr-Weaver T., Szostak J. and Rothstein R., 1981, Proc. Natl. Acad. Sci. USA, 78, pp. 6354-6358). However, the recombination event is rare in practice and only occurs in a few cells. Accordingly, selectable markers are inserted between the sequences bringing about the recombination in order to make it possible, after transformation, to isolate the cells where the integration of the DNA fragment occurred by identifying the markers corresponding, for example, to a resistance to an antibiotic. However, these selectable markers are difficult to eliminate, which has the disadvantage, on the one hand, of not being able to reuse the same marker for another transformation, and, on the other hand, of leaving in the host cell exogenous DNA fragments.
The problem is further complicated for the industrial strains of Saccharomyces cerevisiae which are distinguishable from the laboratory strains by the fact that they are both aneuploid and polyploid, that is to say that many genes are present in several copies: multiple copies in tandem, multiple copies dispersed in the genome, families of genes only slightly different in their sequence and for which no difference in activity has been detected such as the SUC gene for example (Olson M., 1991, Genome Structure and Organization, in Saccharomyces cerevisiae in the Molecular Biology of the Yeast Saccharomycesxe2x80x94Genome Dynamics, Protein Synthesis and Energetics; Ed. Broach, Jones, Pringle, CSHL Press, New York).
This particular ploidy makes any manipulation intended to inactivate a gene by disruption or deletion of its alleles difficult because it is necessary to inactivate all the copies of the gene, most often by repeating the inactivation operation.
The prototrophic character of industrial strains does not make it possible to use so-called xe2x80x9cauxotrophicxe2x80x9d markers and involves only the use of dominant markers in all the transformations, but in this case, it is necessary either to have several different markers, or to eliminate the marker after each transformation so as to be able to reuse it for a new transformation.
Another problem consists in the fact that to carry out any transformation at all the DNA construct used often contains DNA of non-yeast origin which is not always eliminated or not in its entirety. Foreign DNA therefore remains present in the genome of the yeast. However, for marketing in the food industry sector, it is important that the yeasts do not contain DNA not originating from strains belonging to the same genus, preferably to the same yeast species, with the sole possible exception of the part of a gene of interest encoding for a protein which is not naturally produced by the yeast strain, such as for example a xylanase, a malate permease, a malolactic enzyme, a lactate dehydrogenase.
In yeast, the integration of a gene of interest into a DNA fragment or target gene occurs according to the principle of homologous recombination. For that, an integration cassette contains a module comprising at least one yeast marker gene and the gene to be integrated, this module being flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site. These fragments will be termed hereinafter xe2x80x9crecombinogenicxe2x80x9d because they will bring about double homologous recombination allowing the insertion of the cassette. These recombinogenic fragments will be called hereinafter RS (=Recombinogenic Sequence). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences of the construct and the corresponding regions of the target gene results in the inactivation of the target gene caused by the simultaneous integration of the construct, that is to say the replacement of the target gene by the integration cassette.
This technique also applies to the inactivation of a gene, which may be a disruption/interruption or a deletion; in the case of a total deletion, the entire target gene is exchanged; in the case of a disruption/interruption, the sequence of the target gene is interrupted, and in general, it is accompanied by a larger or smaller deletion; accordingly, the terminology xe2x80x9ctotal or partial deletionxe2x80x9d is sometimes used to designate both the deletion and the disruption of a gene, which has the result of blocking or modifying its expression. In these two particular cases, the cassette to be inserted into the target gene may contain only the selectable marker flanked by the recombinogenic homologous fragments.
The problem then posed is that of eliminating the unnecessary exogenous fragments, in particular the markers thus introduced, whose presence is generally considered to be inopportune. For that, it is possible to use the xe2x80x9cpop-outxe2x80x9d or spontaneous excision phenomenon in yeast. It is an intrachromosomal recombination event between identical or similar sequences which can occur naturally. A DNA loop forms between two similar direct sequences, and is then ejected, leaving in place one of the two recombined sequences. The frequency of this phenomenon increasing with the degree of sequence identity, it is possible to promote the elimination of a DNA fragment, for example of a selectable marker, by placing it between two identical direct sequences, termed direct repeat sequences (DRS). Thus, the marker is excised while a direct repeat sequence is conserved.
For example, an article describes a molecular construct (pNKY51) which makes it possible to disrupt or to delete a yeast gene (E. Alani, L. Cao and N. Kleckner, xe2x80x9cA method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strainsxe2x80x9d, Genetics, 116, pp. 541-545, August 1987). The vector is an Escherichia coli plasmid containing the URA3 gene (marker) flanked by direct repeat sequences derived from the Salmonella histidine operon (hisG). This plasmid is digested in order to produce a module (hisG-URA3-hisG) which is then flanked on either side by recombinogenic homologous sequences of the target gene to be disrupted. The cassette thus produced is integrated into the target gene of a Ura31  yeast, and the strains having integrated the cassette may be isolated by selecting the Ura+ strains. After excising the cassette, the strains become Uraxe2x88x92 again.
The construct is used to disrupt or delete the yeast TRP1, SPO13, HO, RAD50 and LEU2 genes. The multiple transformations are carried out either by crossing strains carrying one transformation each, or by a series of transformations with different disruption constructs and by repeated Ura+ and Ura3xe2x88x92 selection cycles in a single strain.
One of the two Salmonella direct repeat sequences hisG remains in the genome after each transformation.
This method makes it possible to reuse the marker (auxotrophic marker) for a new transformation.
This same type of method is described in the document EP 635,574 for transforming bacteria, yeasts or fungi, the essential characteristic of the method being, in place of the URA3 marker, the use of another specific marker gene, the amdS gene. The marker in question seems to be interesting in the case of fungi strains, in particular Aspergillus; however the use of the system appears to be complex in yeasts.
The document EP 220,009 describes a method allowing the integration of total or partial deletion cassettes in yeast using the so-called xe2x80x9cCre-loxxe2x80x9d excision system; this method, developed in animals, can also be used in yeasts; the sequences equivalent to the direct repeat sequences are, in this case, the so-called xe2x80x9cloxxe2x80x9d specific sequences and the excision of the DNA sequence between the two xe2x80x9cloxxe2x80x9d sequences is carried out in the presence of a xe2x80x9cCrexe2x80x9d recombinase.
One of the disadvantages of this system is that one of the xe2x80x9cloxxe2x80x9d sequences which remains is an exogenous DNA from a bacteriophage.
The document EP 814,165 describes a system which makes it possible, here again, to excise a module containing a marker by the action of a specific enzymatic system on sensitive sequences, the system being very similar to the xe2x80x9cCre-loxxe2x80x9d system, insofar as it uses a recombinase specific to the DRS.
The aim of the present invention is to provide yeast transformation cassettes leaving no useless exogenous DNA fragment, and having on the one hand at least one negative and dominant selectable marker ; and on the other hand two direct repeat sequences (DRS) being not exogenous and not recombinogenic with the yeast genome, these 2 direct repeat sequences (DRS) being placed on both sides of the fragment containing the negative dominant marker. This dominant counter-selectable marker is preferably an inducible marker. These DNA cassettes, subject of the present invention, contain preferably a second selectable marker.
The DNA cassette subject of the present invention contains optionally at least one gene of interest, arid additionnally if necessary, the elements required for its expression in the host cell, for example, any appropriate promoter, enhancer and/or terminator. Any exogenous (=heterologous) DNA for the yeast genome, other than that of the said gene(s) of interest, is considered as unnecessary or useless exogenous DNA.
One of the aims of the present invention is to provide an integration/excision cassette allowing the integration of a gene of interest and/or the inactivation of a gene of the host, capable of being used in yeasts, which solves the previous problems, in particular which makes it possible to obtain integrations free of exogenous DNA while leaving in place only the DNA of yeast belonging to the same genus and optionally a gene of interest. In the present description, xe2x80x9cexogenous DNAxe2x80x9d is understood to designate a DNA derived from a genus which is different: or more specifically from a species which is different, from the genus or from the species of the transformed yeast strain.
Accordingly, the present invention relates to a DNA cassette intended for the integration of a gene of interest or for the inactivation of a gene in yeasts, characterized in that it contains:
at least one negative dominant marker, preferably two dominant selectable markers: one positive marker and one negative marker;
two direct repeat sequences (DRS) which are non-exogenous and nonrecombinogenic with the genome of the host strain, these two direct repeat sequences flanking the selectable marker(s);
two recombinogenic DNA sequences (RS) corresponding to the desired insertion site in the yeast, flanking in 5xe2x80x2 and 3xe2x80x2 the abovementioned two direct repeat sequences;
optionally one (or several) gene(s) of interest containing, if necessary, the elements necessary for its expression in the host cell, placed between a DRS sequence and the adjacent recombinogenic DNA sequence (RS).
This integration/excision cassette allows the total or partial deletion of genes present in the genome of industrial yeast strains and optionally the simultaneous integration of a new gene without any foreign DNA fragments remaining in the genome of the strain thus transformed (apart from possibly the newly cloned gene if this is the case). Preferably, the said integration/excision cassette will be designed so as to make it possible to transform several alleles, or even all the alleles of a gene one after the other without the risk of seeing transformations reoccurring in principle at the same site. It is systematically checked that the transformation of all the alleles or of a determined number of them has indeed taken place. The transformation procedure may be repeated several times, by virtue of the use of at least one different recombinogenic sequence (RS) each time, the two RS sequences directing the integration of the cassette. Preferably, the two RS sequences will be changed as explained below; if only one is changed, it will be preferably the RS sequence chosen in 3xe2x80x2 of the insertion site.
When the integration/excision cassette is intended for the integration of a gene of interest, this means that, after the excision, the gene of interest and a DRS sequence will remain, this set being flanked by two recombinogenic sequences; When the cassette is intended for the inactivation of the gene, this may involve the inactivation of a gene by total or partial deletion; in this case, only one DRS sequence flanked by two recombinogenic sequences remains. It is important, in a general way, that the DRS sequence is noncoding; indeed, situated in 3xe2x80x2 of a recombinogenic sequence, it could be expressed. Noncoding sequence is understood to mean a sequence which is not translated in the form of a peptide.
A problem often encountered for the expression of one or several genes of interest at a sufficient level is the need for a high number of copies of the said gene(s) of interest including the elements allowing their expression. Another aim of the present invention is to provide a high copy or expression plasmid for yeast which is free from useless exogenous DNA, which can be obtained by means of a yeast-E. coli shuttle plasmid containing two direct repeat sequences (DRS), being non-exogenous and non-recombinogenic with the genome of the host strain.
This yeastxe2x80x94E. coli shuttle plasmid, preferably S. cerevisiaexe2x80x94E. coli relates also to the invention, its characteristics being that it contains:
at least a negative and dominant selectable marker, preferably two selectable markers,
two non-exogenous and non recombinogenic direct repeat sequences (DRS), these two sequences separating two regions in the circular plasmid: on the one hand the yeast part intended to be kept in the host yeast and to allow the expression of the gene(s) of interest and on the other hand the part or region used for obtaining the shuttle plasmid and intended to be eliminated by excision.
Preferably, the region placed between the two direct repeat sequences and intended to be eliminated by excision contains:
an E. coli replication origin
a selectable marker for E. coli 
the negative dominant marker, preferably inducible, i.e. a gene encoding for a compound which is toxic for yeast, the said gene being expressed under particular conditions of culture medium, corresponding to the induction.
The second region delimited by the two direct repeat sequences (DRS) and intended to become the yeast high copy expression plasmid contains:
a yeast 2 micron replication origin
a selectable marker of yeast origin
an expression system with at least one gene of interest.
Another aim of the invention is also to provide DNA cassette in the form of a yeast high copy plasmid which is obtainable after excision of the hereabove defined yeastxe2x80x94E. coli shuttle plasmid and which contains a DRS sequence being non-exogenous and non-recombinogenic with the genome of the host strain, a yeast 2 micron replication origin, a selectable marker of yeast origin, an expression system with at least one gene of interest.
These new cassettes according to the invention preferably contain two selectable markers, one of those always being thus a negative and preferably inducible dominant marker. A dominant marker for the purposes of the present invention being a marker for prototrophic cells (or non-auxotrophic cells with respect to the protein encoded by the marker) allowing, in a particular growth medium suitable for the marker, only the growth of the clones having the desired modification. This is also called direct selection. A dominant positive marker is a marker whose expression in the host cell ensures survival under certain particular conditions; in most cases, this will be a resistance marker, that is to say that when the corresponding marker is expressed, it allows the cell to be resistant, for example to antibiotics or to other toxic substances. A dominant negative marker is in contrast understood to designate in general a marker whose expression is toxic for the host cells under particular conditions. It may be in particular a marker made xe2x80x9cinduciblexe2x80x9d encoding for a toxic product, such as for example the RTA (Ricin Toxin A) gene, placed under the control of an inducible promoter which is highly repressed by glucose. This inducible promoter may be, for example, a promoter of a GAL gene, or a hybrid promoter such as the GAL10-CYC1 promoter.
The article by Frankel et al., xe2x80x9cSelection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiaexe2x80x9d, Molecular and Cellular Biology, February 1989, pp. 415-420, describes a method of selecting mutants producing an inactive toxin after transformation with a DNA construct containing the gene encoding for the ricin toxin A polypeptide chain under the control of the GAL1 promoter and induction of the expression of this gene.
The use of an inducible gene encoding for a toxic product such as the polypeptide of the ricin toxin A chain according to the present invention is different, because this gene is used as a negative marker allowing a counterselection, that is to say a second selection of the strains transformed by the integration cassette or the high copy plasmid, object of the invention, but which have lost the excision module. Of course, it will be systematically checked that the survival of the transformed yeast cells in an inducible medium is due to the loss of the excision module and not to a mutation of the RTA gene or the gene encoding for another toxic protein, inactivating the toxin produced. This may be carried out for the strains transformed with the integration cassette, on the one hand, by sequencing the transformed zone and, on the other hand, by checking for the absence of any detectable fragment of the RTA gene within the genome of the transformed strain. Similar verifications can be carried out with respect to the loss of certain DNA fragments by the yeast high copy expression plasmids, which are also a subject of the invention.
Examples of positive and negative markers are given below in greater detail.
The presence of two dominant markers makes it possible to provide a system with a very high efficiency and it makes it possible to choose universal markers for yeasts giving an easy direct selection.
It will have been understood that the positive marker makes it possible to directly select the transformed cells and then, after culturing in an inducing medium, the negative marker makes it possible to directly select the cells whose excision module will have been eliminated by xe2x80x9cpop-outxe2x80x9d or excision.
The excision of the module containing the two markers in the integration/excision cassette is carried out by virtue of the presence of the direct repeat sequences (DRS) whose structure also constitutes one of the characteristic features of the present invention.
In the same way, the presence of two markers allows the system constituted by the yeastxe2x80x94E. coli shuttle plasmid to be highly efficient. It will have been understood that the dominant and inducible negative marker allows to select the cells containing only a yeast plasmid or yeast plasmids having excised the part used for the construction of the shuttle plasmid because of to the presence of direct repeat sequences (DRS), characteristic features of the present invention. In this construction, the excision eliminates the negative and preferably inducible dominant marker; whereas the positive marker of yeast origin, which is situated in the other region of the shuttle plasmid delimited by the 2 direct repeat sequences (DRS), is kept and used for keeping a selectable pressure for the maintenance in yeast cells of the yeast high copy expression plasmids, obtained after excision.
These DRS sequences will be preferably chosen from:
yeast mosaic sequences, preferably belonging to the genus Saccharomyces and more preferably Saccharomyces cerevisiae, or
yeast DNA sequences not present in the host strain.
Yeast  less than  less than mosaic sequence  greater than  greater than  is understood to designate a DNA sequence consisting of fragments coming from different DNA sequences of one or more yeast strains, belonging to the same species, or even to the same subspecies.
In order to ensure that the fragments in question lead to the formation of a nonrecombinogenic  less than  less than mosaic sequence  greater than  greater than , each of these fragments will preferably contain less than 30 bp, and the DRS sequence will contain from 80 to 300 bp so as to ensure the excision. The length of the fragments, namely of the order of 10 to 30 bp, does not allow them to play the role of recombinogenic fragments; on the other hand, the overall DRS sequence consisting of these different fragments allows recombination with an identical DRS, and therefore the excision of the DNA fragment between 2 identical DRS sequences. Preferably, a DRS sequence contains about 90 to 200 bp or 210 bp. After excision, one of the 2 DRS sequences remains in place in the genome; it is therefore necessary that this sequence consists of yeast DNA, that is to say of DNA belonging to the same genus and preferably to the same species of yeast.
As was indicated above, it is also possible to use, as DRS sequences, fragments of a yeast gene which is absent from the industrial yeast host strain to be transformed. For example, the Saccharomyces cerevisiae species is described as assimilating and fermenting melibiose in a variable manner depending on the strains. All the industrial strains of bakers"" yeast are melibiose xe2x80x9cminusxe2x80x9d, on the other hand most industrial strains of brewery yeast are melibiose xe2x80x9cplusxe2x80x9d, whereas all these strains belong to the Saccharomyces cerevisiae species. The MEL1 gene is consequently a good candidate for providing the DRS sequences of a transformation cassette of an industrial strain of bakers"" yeast. The MEL1 gene is described in Patent EP 241,044; it is a gene which encodes for the xcex1-galactosidase enzyme which hydrolyses melibiose to galactose and glucose. This gene is absent from the industrial strains of bakers"" yeast which, for this reason, are not capable of consuming the entire raffinose in molasses. In this case, it is possible to use longer DRS fragments, of the order of 200 bp or 210 bp for example, in order to facilitate the excision, but remembering that the DRS sequence remaining after excision should have been made noncoding by any appropriate means such as changing the reading frame or the introduction of stop codons for example.
As already indicated, the DRS sequences described above are flanked in the integration/excision cassette, in 3xe2x80x2 and 5xe2x80x2, by recombinogenic DNA sequences, that is to say which allow integration into the target gene by double homologous recombination and which are called RS sequences. These two DNA fragments are of course prepared from sequences chosen from the target gene which will then correspond to the sites specifically recognized for the homologous recombination. The choice of the RS sequences thus determines the site for insertion of the cassette.
When it is desired to successively integrate the same excision module with optionally a gene of interest into different alleles of the same family of genes, it is necessary to choose the recombinogenic sequences so that they do not become recombined in an allele already transformed.
To do this, successive integration/excision cassettes will be constructed which have at least one recombinogenic sequence which is no longer present in the previous recombined sites, preferably the recombinogenic sequence not present in the previously recombined site will be chosen in 3xe2x80x2 of this site. For example, fragments will be chosen starting from the 3xe2x80x2 end of the target gene for the recombination and then approaching the centre of the said target gene for each successive cassette. Preferably, the 2 RS sequences will be changed each time according to this applied technique by choosing different RS fragments starting from the two ends of the target site for the recombination in the different successive integration cassettes.
The integration marker may be any dominant positive marker, that is to say which allows the yeasts possessing it to survive a selection pressure. This will be preferably a resistance marker, that is to say a gene which confers on the strain resistance towards a toxic component. The use of an auxotrophic marker (gene necessary for growth on a medium lacking a nutritive component) shall be a preferred solution for the maintenance of yeast high copy expression plasmids. It is required that auxotrophic mutations had been introduced into the concerned industrial yeasts by targeted disruption or deletion, like those which have been made possible by the integration/excision cassette according to the present invention.
Among the dominant positive markers which may be used as marker for resistance to a toxic compound, there may be mentioned (the toxic compound being cited first):
Copper: the Saccharomyces cerevisiae CUP1 gene, which allows resistance to copper and to cadmium. Its use as dominant positive selectable marker has been shown in strains sensitive to copper (Henderson et al. 1985, Curr. Genet. 9, pp. 133-138; Pentilxc3xa40 et al. 1987 Curr. Genet. 12, pp. 413-420; Hottiger et al. 1995, Yeast, 11, pp. 1-14).
Cycloheximide: mutant alleles of the Saccharomyces cerevisiae ribosomal CYH2 gene allowing resistance to cycloheximide when they are present with the wild-type CYH2 gene (homozygosity does not confer resistance (Struhl et al. 1983, Gene. 26, pp. 231-242));
FK520: the MDR3 gene, orginating from the mouse, encoding the P-glycoprotein when it is expressed in Saccharomyces cerevisiae confers on it resistance to FK520 (an antifungal and immunosuppressive agent) (Raymond et al., 1994, Mol. Cell. Biol. 14, pp. 277-286);
Fluoroacetate: the dehH1 gene, derived from Moraxella sp. expressed in Saccharomyces cerevisiae allows resistance to fluoroacetate (van den Berg et al., 1997, Yeast, 13(6), pp. 551-559);
Fluorophenylalanine: the dominant AR04-OFP allele, which is a mutation of a nucleotide of the Saccharomyces cerevisiae ARO4 gene, allows resistance to the p-fluoro-DL-phenylalanine, or o-fluoro-DL-phenylalanine, and tyrosine mixture (tyrosine makes it possible to suppress the inhibition of the expression of this gene). Its efficiency as a marker for resistance to an amino acid analogue has been shown on several industrial yeast strains (Shimura et al. 1993, Enzyme Microb. Technol. 15, pp. 874-876);
Formaldehyde: the Saccharomyces cerevisiae SFA1 gene, when it is overexpressed allows resistance to formaldehyde five to seven times higher compared with the corresponding wild-type strain. This gene was used as selectable marker of a multicopy plasmid (Wehner et al. 1993, Yeast, 9, pp. 783-785). One of the advantages of this marker is the low cost of the toxic molecule (van den Berg et al. 1997xe2x80x94Yeast, 13(6), pp. 551-559);
Geneticin: the KanMX module, which contains the coding sequence kanr derived from the Escherichia coli Tn903 transposon, fused with the transcriptional control sequences of the TEF gene of the filamentous fungus Ashbya gossypii, or with any other functional transcriptional terminator and promoter, confers on Saccharomyces cerevisiae resistance to geneticin (G418) (Wach et al. 1994, Yeast, 10, pp. 1793-1808, among the numerous users of this marker);
Glyphosate: the coding part of the Escherichia coli aroA gene, inserted between the Saccharomyces cerevisiae terminator and promoter sequences, allows therein resistance to glyphosate (Kunze et al. 1989, Curr. Genet. 15, pp. 91-98);
Hygromycin B: the hph gene, originating from an Escherichia coli plasmid, placed under the control of the promoter of the Saccharomyces cerevisiae CYC1 gene allows resistance to hygromycin B (Gritz and Davies 1983, Gene. 25, pp. 179-188);
Methotrexate: the Mdhfr gene, encoding a dihydrofolate reductase in mice, confers on Saccharomyces cerevisiae resistance to methotrexate (Zhu et al. 1986, Gene. 50, pp. 225-237);
Phleomycin: the Tn5ble gene, derived from Escherichia coli, allows Saccharomyces cerevisiae to acquire resistance to phleomycin. This gene is generally used with the Saccharomyces cerevisiae CYC1 terminator and TEF1 promoter (Wenzel et al. 1992, Yeast, 8, pp. 667-668);
Sulfometuron: the SMR1-410 gene, mutant allele of the Saccharomyces cerevisiae ILV2 gene, allows resistance to the herbicide sulfometuron, in a dominant manner in diploid heterozygous strains (Xiao et al. 1990, Plasmid. 23, pp. 67-70). A new mutant allele was recently identified (Xie et al. 1996, FEMS Microbiol. Letters, 137, pp. 165-168).
As regards the second marker, namely the negative marker, that is to say the marker for counterselection, it will be, as was indicated above, in most cases a marker made xe2x80x9cinduciblexe2x80x9d and encoding, under induction condition, for a molecule which is toxic for the yeast cell.
An advantageous marker for counterselection is the coding part of the RTA (Ricin Toxin A) gene, placed under the control of an inducible promoter which is both strong and highly repressed by glucose, such as for example the GAL10-CYC1 promoter and a PGK1t terminator. The principle of these constructions from a yeast promoter and terminator is to use a promoter and a terminator derived from different genes, so as to minimize the risks of undesirable recombination in the yeast genome.
Of course it is then checked by sequencing of the transformed region that the exogenous DNA is absent, that is to say that the excision indeed occurred as expected. The absence of any detectable fragment of one or both markers within the genome of the transformed strain is also checked by hybridization, more particularly, the absence of any detectable fragment of the counterselectable marker. In the case of construction of the high copy expression plasmid, the positive marker is kept, and consequently this positive marker must correspond to a non-exogenous or homologous DNA sequence.
Other counterselectable markers may also be chosen among genes that are homologous and heterologous to the yeast genome and whose conditional over-expression on a centromeric plasmid is toxic for the host yeast. For example:
ATL2, gene from Arabidopsis thaliana, coding for a zinc-finger protein (Martinez-Garcia et al. (1996) Gene isolation in Arabidopsis thaliana by conditional overexpression of cDNAs toxic to Saccharomyces cerevisiae: Identification of a novel early response zinc-finger gene. MGG, 252:587-596.)
DUO1, gene from Saccharomyces cerevisiae, coding for a spindle pole body protein (Hofmann et al. (1998) Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function. J. Cell. Bio., 143:1029-1040) ;
GIN11, gene from Saccharomyces cerevisiae coding for a subtelomeric protein (Kawahata et al. (1999) A positive selection for plasmid loss in Saccharomyces cerevisiae using galactose inducible growth inhibitory sequences. Yeast, 15:1-10)
GIN12/SPC42, gene from Saccharomyces cerevisiae, coding for a component of the spindle pole body (Akada et al. (1997) Screening and identification of yeast sequences that cause growth inhibition when over-expressed. MGG 254:267-274)
H1-1, gene from Arabidopsis thaliana, coding for a histone H1 (Martinez-Garcia et al. (1996) Gene isolation in Arabidopsis thaliana by conditional over-expression of cDNAs toxic to Saccharomyces cerevisiae: Identification of a novel early response zinc-finger gene. MGG, 252:587-596.)
TPK1, gene from Saccharomyces cerevisiae, coding for a cAMP dependent protein kinase (Liu, H. et al. (1992) Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose over-expression causes lethality in yeast. Genetics,132:665-673).
The Saccharomyces cerevisiae mosaic repeat sequences correspond, in general, to the construction of a sequence of about 100 base pairs consisting of gene fragments existing in principle in the host cell, each of these fragments being of a length such that it does not in principle allow any recombination event when they are present in a mosaic sequence. These sequences are constructed in a manner such that no peptide chain can be encoded, for example by virtue of the introduction of stop codons at the appropriate sites.
The mosaic sequences may be constructed from short gene fragments which are highly probably conserved in the different yeast strains and are situated in only slightly variable regions of these genes. These only slightly variable regions are determined by searching for homology with other gene libraries using the B.L.A.S.T.A. programme (Altschul et al., 1997, Nucleic Acids Res., 25, pp. 3389-3402). Intron sequences known to be highly variable should be avoided. Preferably, when the host strain belongs to the Saccharomyces cerevisiae species, the choice of the short fragments constituting the mosaic sequence is made based on the sequence of the Saccharomyces cerevisiae strain from the MIPS in Munich (Mewes et al., 1997, Nucleic Acids Res., 25, pp. 28-30). These fragments are also chosen so that the mosaic sequence possesses nonsense codons, stopping the translation in the 6 reading frames (3 direct reading frames and 3 reverse reading frames).
One of the essential characteristics of the excision module existing in the transformation cassette which is the subject of the present invention is that it can be used whichever the chosen integration site in the yeast genome (case of the integration/excision cassette) or whichever the gene(s) of interest to be expressed. This property is directly dependent on the choice of the restriction sites which make it possible to introduce the gene(s) of interest and the RS sequences into the transformation cassette. The presence of a specific restriction site in these DNA fragments to be introduced into the cassette excludes the said site from being used afterwards to construct the said integration/excision cassette. For the excision module to be easily used for any construction, it is necessary that it contains at each of its ends at least one restriction site whose frequency in the genome is as low as possible. One of the characteristics of the invention is that the excision module contains at least 3 rare sites at its ends arid preferably at least 5 rare restriction sites. A rare restriction site is a site which is recognized by a type II endonuclease recognizing an octanucleotide sequence or any restriction site having the same characteristic of rarity. To illustrate this notion of rare frequency, it is recalled that in a DNA sequence having a 50% A-T and 50% G-C composition, the frequency of the presence of a specific hexanucleotide is 1 per 4096 and of a specific octanucleotide is 1 per 65,536. For example, the excision module will have at its 5xe2x80x2 end, the PacI, AscI and PmeI sites, and at its 3xe2x80x2 end the FseI and SwaI sites.
The present invention also relates to a method of integrating a gene of interest or of inactivating a gene in a yeast, characterized in that:
a) the said yeast is transformed with the aid of a DNA construct consisting of the integration/excision cassette as described above,
b) the yeasts having integrated the said cassette are selected by means of a positive marker,
c) and then the yeasts in which the cassette has been excised by virtue of the DRS sequences are selected among these yeasts by searching for the yeasts lacking the negative marker.
More particularly, the invention relates to a method of integrating several copies or of inactivating different copies of a gene in a yeast, characterized in that the method described above is repeated with an integration/excision cassette which contains, for each repetition of the method, at least one different recombinogenic DNA sequence chosen such that each site containing an integration cannot, in principle, be the subject of a recombination with the next cassette.
The integration/excision cassettes optionally containing a gene of interest corresponding to the definitions given above may be constructed according to the customary methods used in the field by persons skilled in the art.
This is also true for the E. coli-yeast shuttle plasmid allowing to obtain, after excision of the E. coli sequences and the negative marker a high copy expression plasmid in the yeast, containing only as exogenous DNA, the exogenous DNA of interest. The principles of construction, the elements of this shuttle plasmid are the same than those here above described.
More particularly, the invention relates to a method of transformation of a auxotrophic yeast in order to obtain many copies of at least one gene of interest with a shuttle plasmid as described above of which the selectable marker or positive marker of yeast origin is a marker complementing the auxotrophy of the host yeast, characterised by:
the said auxotrophic yeast is transformed with the above mentioned shuttle plasmid.
yeast cells, which only contain plasmids, of which the part corresponding to the E. coli fragments and to the negative marker has been excised, are selected on a minimal medium, i.e. a medium which does not contain the element for which the yeast is auxotrophic, this minimal medium being chosen in order to induce the dominant negative marker located in the shuttle plasmid region containing this marker and the E. coli DNA fragments, both flanked by the two DRS sequences.
Among the methods of transformation which can be used for yeast strains, there may be mentioned in particular that proposed by Ito et al. (1983, J. Bacteriol., vol. 153, pp. 163-168), or by Klebe et al. (1983, Gene, vol. 25, pp. 333-341), or by Gysler et al., (1990, Biotechn. Techn., vol. 4, pp. 285-290).
Finally, the present invention relates to the yeasts transformed with a cassette according to the invention and obtained by a method as described above and which contains only yeast DNA, with the possible exception of the gene(s) encoding for a protein of interest, the DRS sequences being noncoding. If the cassette, according to the invention is used for inactivation of a gene family, the yeasts according to the present invention will be such that the desired number of copies, that is to say the desired number of alleles of the same family of genes, will have been inactivated by integration/excision with the aid of cassettes according to the present invention, or optionally the entire copies will have been inactivated, so as to choose the intensity with which a gene will be expressed. Similarly, if one of the cassettes according to the invention is used for the expression of one or several genes of interest, the desired number of copies for the said gene(s) may be obtained.
Among the yeasts which are most particularly advantageous according to the present invention, there may be mentioned the yeasts of the genus Saccharomyces and in particular Saccharomyces cerevisiae, in particular the industrial strains and more particularly those of bakers"" yeasts.